1. Technical Field
This invention relates to determining the genotype of chickens.
2. Description of the Background Art
The poultry breeding business is of major economic importance in the United States and in most parts of the world. Epidemics of viral infectious disease, for example Marek""s disease, in flocks raised for meat or eggs can have a devastating effect to this industry, even in modern facilities. Consequently, development of methods to produce breeding stocks of chickens, whether raised for meat or eggs, which are resistant to disease, is commercially very important.
In chickens, as opposed to most mammals, the particular Mhc haplotypes have readily demonstrated differential influences in the immune response to certain diseases, such as the tumors caused by the highly infectious herpes virus responsible for Marek""s disease. Chickens with different Mhc genotypes respond differently to the infectious pathogen of Marek""s disease, with potentially deadly consequences to animals possessing a relatively unresponsive Mhc genotype (i.e., two non-protective haplotypes). Determining the Mhc genotype of chickens has therefore become important to the poultry industry, so that disease-resistant strains of chickens can be bred.
In domesticated fowl, the known Mhc genes are organized into two separate linkage groups, B and Rfp-Y. FIG. 1 provides a schematic map showing the known chicken Mhc genes. The B system comprises polymorphic classical Mhc class I heavy chain, class II beta chain, B-G genes and other genes. The B system has been known as a highly polymorphic blood group system since the early 1940""s. Rfp-Y was discovered more recently by DNA hybridizations (Briles et al., Immunogenetics 37:408-414 (1993)) and consists of at least two class I heavy chain genes, three class II beta chain genes, a c-type lectin gene and two additional genes of unknown nature. Miller et al., Proc. Natl. Acad. Sci. USA, 91:4397-4401 (1994); Miller et al., Proc. Natl. Acad. Sci. USA 93:3958-3962 (1996).
As with the B region, the Rfp-Y gene region is small. At least one Rfp-Y haplotype contains only a single functionally active class I locus. This suggests that disease associations with particular Rfp-Y haplotypes have a similar basis in a small number of loci. In addition, interactions may occur between alleles of the B and Rfp-Y loci. Particular combinations of haplotypes in the two systems therefore may provide optimal disease resistance for a particular disease.
It has already been observed that when the B system provides intermediate disease resistance to Marek""s disease, the influence of Rfp-Y genotype can be significant. Wakenell et al., Immunogenetics 44:242-245 (1996). This influence may be a direct one wherein the Rfp-Y genes compensate in antigen presentation, however additional interactions could occur between loci in B and Rfp-Y. For example, studies of Mhc Class I loci in mice have shown that antigen presenting molecules have a critical role in controlling the activity of natural killer (NK) cells. Signal peptides cleaved from nascent classical class I polypeptides are presented by at least one non-classical class I molecule and recognized by receptors on NK cells, resulting in modulation of NK cell activity. Natural killer cells are critical in eliminating infected cells in which class I molecule expression has been down-regulated by the infecting pathogen. Having the capacity to detect B and Rfp-Y haplotypes in commercially bred poultry provides a means by which immune responses can be optimized.
In the chicken, the role of particular Mhc haplotypes in disease resistance has been extensively investigated. The influence of the genotype of the Mhc B system and resistance to certain diseases in chickens, for example, Marek""s disease, has been documented by several authors. See Hanson et al., Poult. Sci. 46:1268 (1967); Briles et al., Science 195:193-195 (1977); Briles et al., Science 219:977-979 (1983); Longenecker et al., Immunogenetics 3:401-407 (1976); Dietert et al., Crit. Rev. Poult. Biol. 3:111-129 (1991); Kaufman et al., Immunol. Rev. 167:101-117 (1999). Genotyping of the B complex of chickens, however, has focused mostly on particular lines of White Leghorn birds, a breed raised primarily for egg production. Alloantisera used to determine B haplotypes in particular lines of egg-producing chickens do not work well for B haplotyping in other lines of chickens. This is especially true for those lines used in the production of chickens raised for meat which are genetically somewhat distant from layer lines.
Though the immune response in chickens to Marek""s disease and other viral pathogens is strongly influenced by B complex genotype, other alleles at other loci, including the Rfp-Y gene cluster, perhaps the NK region and other more poorly characterized regions as well, influence Marek""s disease resistance. See Brown et al., Avian Dis. 28:884-899 (1984); Vallejo et al., Anim. Genet. 28:331-337 (1997); Bumstead, Avian Pathol. 27:s78-s81 (1998); Kaufman et al., Avian Pathol. 28:s82-s87 (1998); Bumstead, Rev. Sci. Tech. 17:249-255 (1998); Yonash et al., Anim. Genet. 30:126-135 (1999). Rfp-Y haplotypes differentially influence disease resistance and immunity in chickens. For example, Pharr et al. showed, in chickens of Cornell line N, that with birds homozygous for B system haplotype, skin graft rejection was greater and occurred more quickly when donor and recipient were mismatched for Rfp-Y than when they were Rfp-Y compatible. Pharr et al., Immunogenetics 45:52-158 (1996). Additionally, there is varied evidence for the ability for Rfp-Y differences to stimulate lymphocyte proliferation in vitro (Pharr et al., Immunogenetics 45:52-58 (1996); Juul-Madsen et al., Immunogenetics 45:345 (1997)), indicating that alloresponses to Rfp-Y may be induced.
The products of Rfp-Y genes have a structure similar but not identical to classical class I molecules. The sequence variability inherent in the Rfp-Y class I molecules themselves is sufficient to inherently elicit this type of allogeneic response, but alternatively these molecules could present some form of polymorphic antigens that serve as a minor histocompatibility antigen and produce the described histocompatibility effect. The Rfp-Y loci may be important in providing molecules that supplement the apparently less than comprehensive antigen presentation provided by the B system loci. Mhc-like genes located outside classical Mhc gene regions are implicated in a number of immune response functions in mammalian species, including selection of T-cell population during development. Adachi et al., Proc. Natl. Acad. Sci. (USA) 92:1200-1204 (1995).
The previous work of Wakenell et al. indicates that Rfp-Y haplotypes influence resistance to the commercially important Marek""s disease in the chicken. Studies of Rfp-Y influence on Marek""s disease virus challenge have produced results indicating that Rfp-Y haplotype affects susceptibility to infection in different B complex backgrounds. Wakenell et al., Immunogenetics 44:242-245 (1996). In this study, data comparing incidence of Marek""s disease tumors in chickens carrying three different Y system genes showed that the Rfp-Y system exerts an effect on Marek""s disease resistance and that the influence of Rfp-Y haplotypes in some combinations may be quantitatively similar to that of the B-F region. See Wakenell et al., page 244. Some conflicting data that has been reported might be due to the particular B and Y complex interactions either accentuating or masking the Rfp-Y effects. See Vallejo et al., Anim. Genet. 28:331-337 (1997).
Genes within B and Rfp-Y both have a demonstrated influence in resistance and susceptibility to a number of diseases, including virally-induced tumors, bacterial infections and infections with protozoan parasites. See, for example, Briles et al., Science 195:193-195 (1977); Briles et al., Immunogenetics 20:217-226 (1984); Longenecker et al., Immunogenetics 3:401-407 (1976); Kaufman et al., Hereditas 127:67-73 (1997); Wakenell et al., Immunogenetics 44:242-245 (1996); Vallejo et al., Anim. Genet. 28:331-337 (1997); Lamont, Rev. Sci. Tech. 17:128-142 (1998); Caron et al., Poult. Sci. 76:677-682 (1997); Thacker et al., J. Virol. 69:6439-6444 (1995); Uni et al., Br. Poult. Sci. 36:555-561 (1995); Bacon et al., J. Hered. 86:269-273 (1995); Hlozanek et al., Virology 203:29-35 (1994); Schat et al., Poult. Sci. 73:502-508 (1994); Nakai et al. Avian Dis. 37:1113-1116 (1993); Lamont et al., Immunogenetics 25:284-289 (1987); Cotter et al., Poult. Sci. 77:1846-1851 (1998).
There are additional studies reported in the literature describing the influence of Mhc haplotype in many poultry diseases, for example the regression of Rous sarcoma virus induced tumors, Marek""s disease, infectious laryngotracheitis and coccidiosis. See Yoo et al., Br. Poult. Sci. 33:613-620 (1992); Poulsen et al., Poult. Sci. 73 (Suppl. 1):108 Abstr. (1994); Poulson et al., Poult. Sci. 77:17-21 (1998); Clare et al., Immunogenetics 22:593-599 (1985). Since the association of Mhc haplotype with disease resistance in chickens has been demonstrated, the haplotyping methods described below may be used to select for chickens genetically resistant to a variety of diseases.
One of the most important diseases of poultry, in commercial terms, Marek""s disease, is caused by a highly contagious herpes virus that induces T-cell lymphomas in chickens. The virus exists in poultry-breeding countries throughout the world and is responsible for tremendous losses to the industry. Because of the strong Mhc B influence on survival of infection with Marek""s disease virus, many modern commercial chicken breeders select for or are at least aware of the Mhc B types in their commercial lines. Breeders generally have not been able to test for Rfp-Y genotypes, however.
Vaccination is very effective in reducing losses from Marek""s disease, but vaccine breaks do occur and there is evidence that new, more virulent forms of Marek""s disease virus appear periodically in vaccinated flocks. Importantly, Mhc haplotypes also influence the efficacy of vaccination in commercial flocks, see Bacon et al., Poult. Sci. 73:481-487 (1994); Bacon et al., J. Hered. 86:269-273 (1995); Bacon et al., Avian Dis. 38:65-71 (1994). Genetic resistance is an important adjunct to vaccination in the prevention of Marek""s disease in chickens. Therefore the strategies of selection for beneficial Mhc haplotypes and vaccination may be used together to optimize flock performance. Mhc haplotyping according to this invention may also be used to test for newly-recognized resistant haplotypes so they may be introduced into flocks.
Another disease of consequence in commercially raised chickens is coccidiosis. Coccidiosis is a protozoal disease of poultry and other birds that results in diarrhea, enteritis and weight loss. Coccidiosis occurs everywhere that poultry are raised in large numbers. There are seven valid species of chicken coccidia (Eimeria acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella) that vary in their pathogenicity. Infections with the causative organisms occur most often in young, rapidly growing birds. Administration of anticoccidial drugs in recent years have reduced some losses, however drug resistant forms of coccidia appear to be developing since losses now are increasing despite the extensive use of drugs.
This phenomenon has led to interest in developing alternative means of infection control of this disease based in immunity. Mhc haplotype has been shown to influence resistance, susceptibility and immunity to Eimeria. See for example, Caron et al., Poult. Sci. 76:677-682 (1997), Brake et al., Infect. Immun. 65:1204-1210 (1997); Nakai et al., Avian Dis. 37:1113-1116 (1993). Mhc haplotype differences are correlated with differences in caecal lesion scores and weight gain during infection. Also, just as with Marek""s disease, Mhc haplotype influences the effectiveness of immunizations. Methods of chicken haplotying therefore can be used advantageously to select birds resistant to coccidiosis or with improved immune response to Eimeria ssp. upon vaccination.
Another acute viral disease of commercial importance is laryngotracheitis. This disease currently is managed by strict separation of susceptible flocks and by vaccination. Particular Mhc B haplotypes have been found to differ significantly in their influence in laryngotracheitis. As with Marek""s disease, laryngotracheitis is caused by a herpes virus and immune responsiveness apparently is a component of susceptibility to this disease as well. Again, as with Marek""s disease, Mhc haplotype influences the efficacy of vaccination against laryngotracheitis (birds of some haplotypes require higher dosage of vaccine to achieve protection). Poulsen et al., Poult. Sci. 73 (Suppl. 1):108 Abstr. (1994); Poulson et al., Poult. Sci. 77:17-21 (1998).
Genes located within chicken Mhc regions have significant effects on the immune response to pathogens that can be detected experimentally. For example, the capacity of chickens to regress tumors caused by avian leukosis virus is associated with the capacity of T cells to respond to the presentation of Mhc restricted antigen. Thacker et al. J. Virol. 69:6439-6444 (1995). A number of the standard and recombinant B haplotypes have been categorized as either progressor or regressor haplotypes. Brown et al. Immunogenetics 19:141-147 (1984); Collins et al. Poult. Sci. 64:2017-2019 (1985); Taylor et al. Anim. Genet. 19:277-284 (1988); Lukacs et al. Poult. Sci. 68:233-237 (1989); Aeed et al. Anim. Genet. 24:177-181 (1993); White et al. Poult. Sci. 73:836-842 (1994). In the Rous sarcoma virus experimental system, immunity is v-src-specific. Gelman et al., Cancer Res. 53:915-920 (1993); Plachy et al., Immunogenetics 40:257-265 (1994). There is evidence that B haplotype is also associated with shedding of avian leukosis group-specific antigen and hence may influence susceptibility to post-hatching infection from other infected birds. Yoo et al., Br. Poult. Sci 33:613-620 (1992).
Further associations between Mhc haplotype and resistance to two bacterial pathogensxe2x80x94fowl cholera and salmonella are reported. Lamont et al., Immunogenetics 25:284-289 (1987); Cotter et al., Poult. Sci. 77:1846-1851 (1998). These reports demonstrate the importance of Mhc haplotype to immunity in chickens against several commercially important diseases and to the important experimental model, Rous sarcoma virus, and suggest that genetic selection for particular Mhc haplotypes is valuable to breeders for the production of both individuals and flocks that are resistant to numerous diseases.
Selection for B haplotypes providing resistance to Marek""s disease is performed by a number of companies breeding chickens for the production of eggs. Generally, selection is done on the basis of the results of hemagglutination assays using alloantisera that have been developed for particular breeding lines within the company""s flocks. These serological typing methods can be applied to birds within a population once appropriate serological reagents have been developed, however alloantisera made in one population are usually not useful to type other populations. See Li et al., Immunogenetics 49:215-224 (1999). Because most of the alloantisera currently available were prepared for chickens bred for eggs (primarily the White Leghorn breed), there are few reagents available for haplotyping other breeds of chickens.
Development of appropriate alloantisera is a lengthy procedure, generally requiring several years. In addition, the genetic background of the birds, including at least some information with respect to other blood group systems, should be known before the alloantisera are produced. This requirement poses a major disadvantage. In the past, the genetics of birds used as donors and recipients in the immunizations to produce alloantisera have been surmised by initial approximations of the genetic differences using alloantisera from other flocks. The alloantisera specific for a particular flock must be made by reciprocal immunizations between sire and dam in fully pedigreed stock, and then tested by hemagglutination assay among the fully pedigreed progeny of the birds that served as donors and recipients in the immunizations. Cross-reactivity among B haplotypes is commonly encountered, necessitating appropriate adsorptions of the sera to enhance their specificity for the individual Mhc B haplotypes. Because any alloantiserum potentially contains antibodies to a number of polymorphic cell surface markers, considerable care must be taken in typing poorly characterized flocks. Accurate results require considerable attention to detail.
The existing serological reagents from egg-producing chickens are not useful in other chicken breeds. Mhc marker assisted selection for Marek""s disease resistant broiler chickens is not performed routinely, in part because of the lengthy effort needed to develop typing methods based on alloantisera and in part because of the breeding methods used to maintain broiler breeder stock. Therefore, a simple method for Mhc haplotyping for these birds is not currently generally available. No serological reagents exist for the Rfp-Y system in any breed of chicken. The B system and the Rfp-Y system of chickens of all breeds, even those not belonging to the White Leghorn breed, can be studied advantageously using the inventive methods and probes, allowing Mhc marker assisted selection to be applied in selecting for additional disease resistance in breeding stock.
DNA-based typing methods, although currently more expensive on a per test basis, have obvious advantages in that nucleotide probes can be used to determine Mhc haplotypes in flocks without the enormous investment of time and labor required to make alloantisera. One such method relies on the patterns of B-G gene restriction fragments revealed in genomic DNA digested with a restriction enzyme and analyzed by Southern hybridization with nucleic acid probes for the B-G genes. See Miller, U.S. Pat. No. 5,451,670. An advantage of this type of approach is that prior knowledge of gene sequences is not necessary. Another method relies on gene restriction fragment patterns revealed in genomic DNA digested with several restriction enzymes and analyzed by Southern hybridization with non-system-specific nucleic acid probes for the B-F and B-L genes. See Lamont, S. J. et al., Poult. Sci., 69:1195 (1990). Yet another similar method is based on hybridization of oligonucleotide probes specific for known sequences in the various alleles of the B system class I gene. See Heath et al., Poult. Sci. 73(Suppl 1):5 (1994).
Various applications of Southern hybridization with B system probes have been reported in the literature. See Chausse et al., Immunogenetics 29:127-130 (1989); Goto et al., Immunogenetics 27:102-109 (1988); Miller et al., Immunogenetics 28:Z374-379 (1988); Briles et al., Immunogenetics 37:408-414 (1993); Pharr et al., J. Hered. 6:504-512 (1997). The B-G gene probes which are useful in ascertaining B haplotypes because of their close linkage to B class I and class II loci are often sufficient in known stocks of birds for the assignment of B haplotype. The B-F and B-L probes are useful in revealing polymorphic restriction fragment patterns, but they show cross hybridization (recognition) with genes both in B and Rfp-Y gene clusters since each of the B-F and B-L probes were developed without knowledge of sequence differences in the B and Rfp-Y genes.
Because the class I and class II genes in Mhc B and Rfp-Y are fairly closely related, probes for the B system crosshybridize to varying degrees with Rfp-Y genes. It therefore is difficult to use these methods to type birds for polymorphisms in either system in the presence of polymorphisms that are contributed by the other system. For this reason, the probes initially used to identify the Rfp-Y cluster were B system probes able to hybridize to genes in both the Rfp-Y and B gene clusters. Because of the crossreactivity, these types of tests often cannot provide useful Rfp-Y data unless analysis is performed on fully pedigreed families of birds and B-G typing is also performed. Otherwise it is not possible to distinguish which restriction fragments result from each system. Indeed, the presence of Rfp-Y was only found because fully pedigreed animals happened to be the subject of a study with another objective.
DNA-based Mhc typing based on specific sequences may be used, however one must have some sequence data for genes within each haplotype in the population to be tested. This requirement is a major stumbling block to development of an easy, comprehensive haplotyping method for B and Rfp-Y system genes. It is difficult, at least initially, to obtain complete haplotype information about a particular bird using these methods without making sequence determinations for each allele at each locus chosen to represent the entire haplotype.
The use of a technique known as polymerase chain reaction, single-stranded conformational polymorphism (xe2x80x9cPCR-SSCPxe2x80x9d) has been proposed to study the expression of genes in non-erythroid tissues. Miller, M. M. and Goto, R. M., Avian Immunology in Progress, Tours (France), Aug. 31-Sep. 2, 1993, Ed. INRA, Paris 1993 (Les Colloques, No. 62); Zoorob et al., PCT/FR98/02501. In this method, short segments of genes of interest are amplified using the PCR. The PCR products are then heat denatured and applied to a non-denaturing polyacrylamide gel. The single-stranded fragments of the heat-denatured DNA fragments assume secondary conformations determined by their sequences and migrate differently in the polyacrylamide gel during electrophoresis, producing a pattern (or fingerprint) representative of the sequences within the genome in the region of amplification. For this method, oligonucleotide primer sets that hybridize to conserved sequence sites surrounding the polymorphic regions must be developed for the different alleles to be typed. Therefore, a certain amount of knowledge regarding the structure of the genes to be studied is required. PCT application PCT/FR98/02501 discloses methods of detecting Mhc genes in birds such as chickens which are related to resistance to virally-induced tumors, for selection of animals having a desired genotype. Specific nucleic acid probes are disclosed which are able to discriminate between genes of the B and Rfp-Y systems.
Currently, there are no commercially available tests to determine the haplotype in the Rfp-Y system. There are no alloantisera. Consequently, a test which would allow breeders, researchers, and others to rapidly determine the haplotype of birds using relatively straightforward techniques is needed. An ideal test would be quick, simple to perform, and avoid the need for specialized equipment beyond that commonly found in a molecular biology laboratory. The test would not require alloantisera which might not be available for use in all birds or detailed knowledge of the genetics of the birds to be tested. Such a test which could determine the haplotype in the Rfp-Y system as well as the B system using a single set of reagents for each system would be highly desirable, and could be used to aid in breeding birds with increased resistance to disease.
Accordingly, the present invention provides the probes of SEQ ID NO: 1 and 2, and probes which contain at least about 17 consecutive nucleotides of these sequences and are about 17 to about 1,000 nucleotides in length. Preferred probes are about 100 to about 1,000 nucleotides in length. Most preferred probes are those of SEQ ID NOS: 1 and 2. Probes which are fragments of SEQ ID NOS: 1 and 2 are contemplated by the invention, from about 17 nucleotides to one nucleotide less than the entire sequence. Probes which are at least about 70% homologous, or preferably at least about 90% homologous to SEQ ID NOS: 1 and 2 are also provided. Because of the nature of DNA hybridization, higher degrees of homology are required for shorter probes; e.g., only a perfect match or a single nucleotide mismatch is preferred for probes of minimum (about 17 nucleotides) length.
The invention provides methods for breeding chickens to produce disease-resistant offspring by selecting a disease-resistant chicken for mating using these probes. The method involves providing a genomic DNA sample from at least one chicken, digesting the sample with one or more restriction endonucleases to obtain restriction fragments and resolving the restriction fragments, preferably by electrophoresis. The resolved fragments are then optimally transferred to one or more hybridization membranes and optionally immobilized there. The resolved fragments are then incubated with a labeled probe as described above such that the probe hybridizes. Unhybridized probe is removed and an image of the labeled hybridized probe is created, to form a restriction fragment pattern. From this restriction fragment pattern, the Mhc genotype of the chicken providing the DNA sample is determined. If desired, the probe can be stripped and a second probe used in the same manner to create a second restriction fragment pattern.
In a preferred embodiment, the resolved restriction fragments from the genomic DNA sample of a single bird are probed twice, once with a probe specific for the Rfpxe2x80x94Y system and one specific for the B system of the chicken Mhc. Most preferably, the probes of SEQ ID NOS: 1 and 2 are used sequentially or on parallel samples of genomic DNA from the same chicken. Once the Mhc genotype of a chicken has been determined, the genotype is correlated with disease-resistance and a chicken having an Mhc genotype which correlates with disease-resistance is selected for mating. The selected chicken is mated with a second chicken of opposite gender. Preferably, the second chicken has also been selected for a Mhc genotype correlating with disease resistance according to the invention. The invention also provides methods for selecting chickens which are disease-resistant as described above, and methods for determining the Mhc genotype of chickens as described above.